Project Summary Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. It is inherited through mitosis and has roles in transcriptional silencing, centromere specification and genome integrity, which profoundly impact epigenetic mechanisms in health and disease. We have found that the epigenetic inheritance of heterochromatin in the fission yeast S.pombe requires RNA interference (RNAi) to guide histone modification, which occurs during the DNA replication phase of the cell cycle. S.pombe centromeric repeats have an alternating arrangement of small RNA clusters and origins of replication that makes collision of the transcription and replication machineries all but inevitable. We found that RNAi promotes release of RNA polymerase (Pol II) during S phase, allowing completion of DNA replication by the leading strand DNA polymerase, which recruits the histone-modifying Rik1 complex to spread heterochromatin along with DNA replication. In the absence of RNAi, stalled forks are repaired by homologous recombination (HR) without histone modification, so that HR is essential in the absence of RNAi, and reduces the copy number of rDNA repeats. This model may explain the participation of non-coding RNA and DNA repair in many examples of epigenetic silencing, such as imprinting and X-inactivation. Recently, we have found that RNAi is essential for viability in quiescent cells (G0), which are predominant in yeast and play critical roles in cancer, stem cells and neuronal disease. Genetic screens have revealed that the Rik1 complex and Pol II are both involved in this novel function for RNAi, implicating both heterochromatic silencing and DNA repair. S.pombe is an outstanding model system for cell cycle research, heterochromatic silencing, and RNAi. We will examine the roles of DNA replication, RNA Polymerase release, DNA recombination and repair in heterochromatic histone modification mediated by the Rik1 complex and RNAi.